1. Field of the Invention
This invention relates to an improved dyed amylose for .alpha.-amylase assays and more particularly to an improved dyed amylase for the analysis of plant .alpha.-amylases.
2. Description of the Prior Art
Chromogenic substrates for the determination of amylase activity are not new to the art. Generally, they are been prepared by reacting amylose with Cibachron Blue F3GA (a monochlorotriazine dye with aminobenzene sulfonate on one carbon atom and amino anthoquinone sulfonic acid on a second carbon atom and has a molecular weight of 773.5), sodium sulfate and trisodium phosphate. Although the order of introducing the reagents varies depending on the person preparing the substrate, a widely used procedure is described by Klein et al, Anal. Biochem. 31, 412-425, 1969. An aqueous suspension of amylose is warmed and treated with an aqueous solution of Cibachron Blue F3GA. Sodium sulfate is added gradually followed by a warm aqueous solution of trisodium phosphate. After heating and stirring the mixture for about 75 minutes, the dyed amylose is allowed to settle and the supernatant fluid is removed. The residue is resuspended in water with vigorous stirring, allowed to settle, and the supernatant fluid removed. The residue is then washed with methanol, air-dried, and milled to 60-200 mesh. The dyed-amylose substrate was then used at a level of 200 mg/assay to determine amylose activity in human serum and saliva, crystalline hog pancreatic .alpha.-amylase, and fungal amyloglucosidase. Ewen, Clinica Chimica Acta 47, 233-245, 1973, prepared a dyed amylose substrate using the same reagents that Klein et al used but varied the order in which the reagents where combined. Although a tenfold increase in sensitivity was claimed for this preparation, each assay still required the use of 200 mg of substrate for the determination of amylose activity in human serum and in 24 hour urine specimens.
Although, as described above, chromogenic substrates have been widely used for .alpha.-amylase assays in the medical and clinical fields, the commerically available chromogenic substrates have not been very suitable for analysis of plant .alpha.-amylases. Dyed amylopectin has been used to determine .alpha.-amylases in sweet potatoes (J. Food Sci. 38, 548, 1973). However, at 40.degree. C. incubation, extrapolation to "zero" enzyme concentration gave an absorbance of about 0.03 units (625 nm), and the linear portion at 40.degree. C. extended only to 0.225 absorbance units. At 60.degree. C. linearity was observed only up to 0.45 (595 nm).